Project Summary Introduction: We propose to Identify the mechanisms by which an intestinal Th17 immune response contributes to severe and recurrent Clostridioides difficile infection (CDI) and explore the immune mechanism by which fecal microbiota transplant (FMT) protects. Hypothesis: Th17 cells contribute to severe CDI and to recurrence. Premise: Failure of antibiotic treatment of CDI (i.e. death or recurrence) is due to a gut Th17 immune response. Significance: C. difficile is the leading cause of hospital-acquired infection in the United States. One in five patients with CDI fails antibiotic treatment and as a result suffers a recurrent infection or death. This proposal will explore if immunotherapy would improve treatment of CDI, specifically by testing the importance of gut Th17 immune responses in CDI severity and recurrence Investigators: Dr. William Petri at the University of Virginia has discovered that Type-17 immunity causes more severe disease during CDI. This has included showing that IL-23 knockout mice have increased survival, reduced neutrophil influx, and reduced tissue pathology (Buonomo et al 2013), that the Th17 polarizing cytokines IL-6 and IL-23 are associated with more severe disease in humans, that type 2 immunity protects by countering Th17 (Frisbee et al 2019), and that adoptive transfer of Th17 cells is sufficient to enhance CDI mortality (Saleh et al 2019). He is joined by Dr. Ann Hays who directs the complicated C. difficile clinic at UVA and the biostatistical expertise of Dr. Jennie Ma. Innovation: The proposed research by testing if Th17 cells have a role in severe CDI as well as recurrent CDI will be paradigm shifting, as this is a field of research that in the past has focused on targeting the bacterium or the microbiota for therapy. This proposal will instead test if the induction of a Th17 immune response in addition to the bacterium and microbiota are causing disease. Approach: Specific Aim 1: SEVERE CDI - Identify the mechanisms by which a Th17 immune response leads to severe C. difficile infection (CDI) Specific Aim 2: RECURRENT CDI - Test the role of Th17 cells and the Th17-recruiting chemokine CCL5 in recurrent CDI Specific Aim 3: FMT ? Determine if FMT protects from primary and recurrent CDI by inducing IL-33 that blocks the action of Th17. Environment: Key to success are the complementary expertise of the Petri lab in the mucosal immunology of enteric infections, and the clinical expertise in FMT for complicated C. difficile infection of Dr. Ann Hays.